Optimized promoter PyqjfC55 and application thereof in preparation of high-temperature-resistant microbial sensor

Abstract

The invention discloses an optimized promoter PyqjfC55 and an application of the optimized promoter PyqjfC55 in preparation of a high-temperature-resistant microbial sensor. A promoter yqjfC55 is mutated by an error-prone PCR (Polymerase Chain Reaction) method to obtain an optimized promoter PyqjfC55, the optimized promoter PyqjfC55 is connected with yellow fluorescent protein EYFP through fusion enzyme, and finally, the high-temperature-resistant and high-sensitivity microbial sensor capable of sensing explosive molecules 2, 4-DNT in a high-temperature environment is obtained. The optimal culture temperature of the Escherichia coli host is 37 DEG C, the effective working temperature is 42 DEG C, and the Escherichia coli host has the capability of maintaining metabolic activity in a high-temperature detection environment. The biosensor provided by the invention realizes high-sensitivity detection of explosive molecules in a high-temperature environment, improves the sensitivity of the detector for detecting the explosive molecules, and expands the application scene.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and molecular biology, and specifically relates to an optimized promoter PyqjfC55 and application thereof in preparing a high temperature resistant microbial sensor. Background Art

[0002] Explosives (such as TNT, RDX, 2,4-DNT, etc.) pose a serious threat to public safety and ecological health due to their high toxicity and widespread environmental residues. Although traditional detection methods (such as gas chromatography and mass spectrometry) are highly accurate, they rely on complex pre-treatment processes and expensive equipment, and are difficult to meet the needs of rapid on-site detection. Therefore, the development of efficient and portable detection technologies has become an urgent need. Microbial sensors are biosensors that combine living microorganisms with signal conversion elements. The core principle is to use the metabolic activity or specific recognition ability of microorganisms to convert the presence of target substances into detectable electrical or optical signals. The development of this type of sensor began in the 1960s. With the advancement of immobilization technology, the stability and sensitivity of microbial sensors have been significantly improved, and they have gradually been used in environmental monitoring, medical diagnosis, and industrial process control.

[0003] The advantages of microbial sensors lie in their high selectivity and environmental adaptability. For example, some microorganisms can generate electrochemical signals by metabolizing specific compounds (such as nitroaromatic explosives), or enhance the specific response to target molecules through genetic engineering. In addition, microbial sensors have the characteristics of low cost, simple operation, and real-time monitoring, which makes them have great application potential in complex environments. However, at present, microbial sensors are limited by the living environment of microorganisms and therefore cannot meet the needs of some extreme environments. Summary of the invention

[0004] The purpose of the present invention is to provide an optimized promoter PyqjfC55 and its application in preparing a high temperature resistant microbial sensor. The present invention first optimizes the original yqjfC55 promoter to obtain a high temperature resistant promoter PyqjfC55, and uses the optimized promoter and yellow fluorescent protein EYFP to prepare a biosensor that can detect explosive molecules under high temperature conditions, thereby realizing rapid fluorescence detection of explosives under high temperature conditions.

[0005] In order to achieve the above-mentioned invention object, the present invention adopts the following technical solutions: The present invention provides an optimized promoter PyqjfC55, whose nucleotide sequence is shown in SEQ ID NO.3.

[0006] The present invention provides the use of the optimized promoter PyqjfC55 in preparing a high temperature resistant microbial sensor.

[0007] Furthermore, the application comprises the following steps: (1) The yellow fluorescent protein EYFP and the promoter PyqjfC55 were amplified by PCR, and the two fragments EYFP and PyqjfC55 obtained by purification were connected with the plasmid pACYC. The ligation products were transformed into Escherichia coli competent cells, and positive clones were screened on LB solid plates containing antibiotics to obtain the recombinant plasmid pACYC-PyqjfC55-EYFP; (2) The recombinant plasmid pACYC-PyqjfC55-EYFP was chemically transformed into Escherichia coli competent cells and screened on LB solid plates containing 12.5 μg / mL tetracycline resistance at 37°C. The obtained positive clones were introduced into Escherichia coli cells to obtain a high-temperature resistant microbial sensor containing the optimized promoter PyqjfC55.

[0008] Furthermore, the mass ratio of the fragment EYFP and the fragment PyqjfC55 to the plasmid pACYC is 1:1:5.

[0009] Furthermore, the nucleotide sequence of the promoter yqjfC55 is shown in SEQ ID NO.1.

[0010] Furthermore, the nucleotide sequence of the yellow fluorescent protein EYFP is shown in SEQ ID NO.2.

[0011] Furthermore, the nucleotide sequence of the optimized PyqjfC55 promoter is shown in SEQ ID NO.3.

[0012] The present invention also provides the application of the high temperature resistant microbial sensor to detect explosive molecules in a high temperature application environment.

[0013] Furthermore, the method for using the microbial sensor is: mix the high temperature resistant biosensor and the sample to be tested in a volume ratio of 8-10:1, incubate at 40-42°C, take samples once every hour for a total of ten hours, and use an enzyme marker to detect the fluorescence intensity value. The higher the concentration of the sample to be tested, the higher the fluorescence intensity.

[0014] Furthermore, the sample to be tested is the explosive molecule 2,4-DNT, and its concentration is 0.1 mg / L and above.

[0015] Furthermore, the detection concentration range of 2,4-DNT was 0.1 mg / L~10 mg / L.

[0016] Furthermore, the high temperature application environment includes deserts, mountains, and grasslands.

[0017] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. The present invention molecularly transforms the specific promoter of Escherichia coli to make it highly tolerant and sensitive in extreme high temperature environments. The present invention uses Escherichia coli as a host cell, the optimal growth temperature is 37°C, and it can stably exist at a high temperature of 42°C.

[0018] 2. The present invention uses a plasmid containing a mutated PyqjfC55 promoter and yellow fluorescent protein EYFP as a basic plasmid to prepare a biosensor for detecting explosive molecules under high temperature conditions. Therefore, compared with commonly used microbial sensors, the biosensor of the present invention can quickly and sensitively detect explosive molecules under high temperature conditions and has a good application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the plasmid map of the constructed vector pACYC-PyqjfC55-EYFP.

[0020] Figure 2 This is the result of yellow fluorescence screening of mutant strains.

[0021] Figure 3 Yellow fluorescence detection results of the constructed engineered strain.

[0022] Figure 4 Schematic diagram of the mutation site of the optimized promoter PyqjfC55. DETAILED DESCRIPTION

[0023] The present invention is further described below in conjunction with specific embodiments, but the present invention is not limited by the embodiments.

[0024] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased.

[0025] Example 1: Gene acquisition and vector construction 1. Acquisition of genes The yellow fluorescent protein EYFP, whose nucleotide sequence is shown in SEQ ID NO.2, was synthesized by GENEWISE Corporation into a universal vector pUC-Amp vector to obtain a pUC-EYFP vector.

[0026] 2. Construction of pACYC-yqjfC55-EYFP expression vector (1) Using pUC-yqjfC55 plasmid (cited from plasmid p-yqjF in patent CN 113005070B) as a template, primers C55-F and C55-R, polymerase chain reaction (PCR) was performed to amplify the yqjfC55 promoter fragment. The PCR amplification system was as follows: template 1 μL; primer C55-F 2 μL; primer C55-R 2 μL; 2 × Phanta Max Master Mix (Dye Plus) (Vazyme) 25 μL; ultrapure water was added to 50 μL.

[0027] The PCR amplification program was: 95ºC for 3 min; 30 cycles × (95ºC for 15 s, 61ºC for 15 s, 72ºC for 30 s); 72ºC for 5 min; 16ºC ∞.

[0028] The primer sequences are as follows: C55-F: 5'-GTTTTTTTACCTCCTGAGCTCGCCACTCAGGCTGCTGATT-3' (SEQ ID NO. 4); C55-R: 5'-GGGATCCCCGGGTACCCGGTTTTGGCGTAT-3' (SEQ ID NO. 5); The PCR products were purified by gel recovery using a gel recovery kit (Vazyme).

[0029] (2) Using pUC-EYFP as a template, primers EYFP-F and EYFP-R, polymerase chain reaction (PCR) was performed to amplify the EYFP fragment. The PCR amplification system was as follows: template 1 μL; primer EYFP-F 2 μL; primer EYFP-R 2 μL; 2× Phanta Max Master Mix (Dye Plus) (Vazyme) 25 μL; ultrapure water to 50 μL.

[0030] The PCR amplification program was: 95ºC for 3 min; 30 cycles × (95ºC for 15 s, 57ºC for 15 s, 72ºC for 30 s); 72ºC for 5 min; 16ºC ∞.

[0031] The primer sequences are as follows: EYFP-F: 5'-CTCACTATAGGGCGAATTCGAATTACTTGTACAGCTCGTCCATGCC-3' (SEQ ID NO. 6); EYFP-R: 5'-AGGAGGTAAAAAAACATGGTGAGCAAGGGC-3' (SEQ ID NO. 7).

[0032] The PCR products were purified by gel recovery using a gel recovery kit (Vazyme).

[0033] (3) Clone the yqjfC55 and EYFP fragments into the pACYC plasmid using seamless cloning: The two fragments EYFP and yqjfC55 were fused with the plasmid pACYC at a mass ratio of 1:1:5 using the seamless cloning enzyme 2 × ClonExpress Mix (Vazyme).

[0034] The ligation system was incubated at 50 ºC for 30 min. E. coli DH5α competent cells were plated on LB solid plates containing 12.5 mg / L tetracycline resistance, positive clones were screened by PCR, and recombinant plasmid pACYC-yqjfC55-EYFP was extracted from the positive clones, and then positively screened by PCR and identified by sequencing.

[0035] Example 2: Sensor plasmid acquisition The recombinant plasmid pACYC-yqjfC55-EYF was transformed into Escherichia coli MG1655 The competent cells were spread onto LB solid plates containing 12.5 mg / L tetracycline resistance, and positive clones were obtained by PCR screening, thereby obtaining the pACYC-C55-EYFP plasmid and engineered bacteria G4.

[0036] Example 3: Molecular modification and optimization of the promoter yqjfC55 gene to improve the high temperature tolerance and sensitivity of the microbial sensor 1. Error-prone PCR to obtain the mutant PyqjfC55 promoter gene fragment The following primers were used to amplify the yqjfC55 promoter gene in the plasmid pACYC-yqjfC55-EYFP. The yqjfC55 promoter gene was mutated by using a low-fidelity enzyme to change the base pairs during amplification. The PCR amplification system is as follows: Template 1 1 μL; C55-F 2 μL; C55-R 2 μL; 2 × Rapid Taq Master Mix (Vazyme) 25 μL; ultrapure water to 50 μL.

[0037] The PCR amplification program was: 95ºC 3 min; 10 cycles × (95ºC 15 s, 60ºC 15 s, 72ºC 5 min); 72ºC 5 min; 95ºC 3 min; 30 cycles × (95ºC 15 s, 59ºC 15 s, 72ºC 2 min); 72ºC 5 min16ºC ∞.

[0038] The primer sequences are as follows: C55-F: 5'-GTTTTTTTACCTCCTGAGCTCGCCACTCAGGCTGCTGATT-3' (SEQ ID NO. 4); C55-R: 5'-GGGATCCCCGGGTACCCGGTTTTGGCGTAT-3' (SEQ ID NO.5); the obtained PCR product was purified using the Vazyme FastPure Gel DNA Extraction Mini Kit (Vazyme).

[0039] 2. Using the pACYC-yqjfC55-EYFP plasmid as a template, primers C55-RF and C55-RR, polymerase chain reaction (PCR) was performed to amplify the pACYC-EYFP framework. The PCR amplification system was as follows: template 1 μL; primer 1 2 μL; primer 2 2 μL; 2 × Phanta Max Master Mix (Dye Plus) (Vazyme) 25 μL; ultrapure water was added to 50 μL.

[0040] The PCR amplification program was: 95ºC for 3 min; 30 cycles × (95ºC for 15 s, 61ºC for 15 s, 72ºC for 5 min); 72ºC for 5 min; 16ºC ∞.

[0041] C55-RF: 5'-GTTTTTTTACCTCCT-3' (SEQ ID NO.8); C55-RR:5'-GTACCCGGGGATCCCGAT-3'(SEQ ID NO.9) The obtained PCR product was purified using the Vazyme FastPure Gel DNA Extraction Mini Kit (Vazyme).

[0042] 3. Use seamless cloning to clone the mutant PyqjfC55 fragment into the pACYC-EYFP plasmid. The system is as follows: 80 ng of plasmid digestion fragment; 200 ng of PCR product fragment; 5 μL of 2 × ClonExpress Mix (Vazyme); ultrapure water to 10 μL. Incubate the ligation system at 50 ºC for 30 min. Transformation of ligation product E. coli MG1655 The competent cells were plated on LB solid plates containing 12.5 mg / L tetracycline resistance.

[0043] Example 4: Screening of the thermostable PyqjfC55 promoter 1. Strain activation and cultivation A single colony on the plate was picked and transferred to a 48-well plate containing LB liquid culture medium containing 12.5 mg / L tetracycline resistance, with one single colony in each well, and cultured at 37° C. overnight to obtain mutant engineered bacteria.

[0044] 1% of the bacterial solution was transferred to a 48-well plate containing M9 liquid culture medium containing 12.5 mg / L tetracycline resistance and cultured to OD 0.2; 10 mg / L 2,4-DNT was added to the bacterial solution in the 48-well plate and cultured in a shaking incubator at 42°C.

[0045] 2. Prepare 2,4-DNT solution Prepare 2 mg / mL stock solution of 2,4-DNT (10 mg 2,4-DNT dissolved in 5 mL anhydrous ethanol); Prepare the diluted 2,4-DNT solution in the following proportions: 10 mg / L: 5 μL stock solution + 980 μL M9 medium + 15 μL anhydrous ethanol; 3. Luminescence detection The fluorescence intensity of each sample was detected by an ELISA reader (Biotek) after 3 h, and the mutant engineered strains G1, G2, G3, and G4 with the largest fluorescence difference were screened.

[0046] The results are as follows Figure 2 The results showed that among the mutant strains G1, G2, G3, and G4, G1 had the best effect under the induction of 10 mg / L 2,4-DNT concentration at 42°C and formed a significant difference with the control group G1. At the same time, there was a significant difference between the presence of 10 mg / L 2,4-DNT explosive molecules and the absence of 2,4-DNT explosive molecules. Thus, the high temperature resistant microbial sensor G1 was obtained.

[0047] Example 5: Application of microbial self-luminous biosensor to detect explosive molecules under high temperature conditions 1. Strain activation and cultivation The correctly sequenced engineered strain G1 was transferred to LB liquid culture medium containing 12.5 mg / L tetracycline and cultured at 37°C overnight to obtain a bacterial solution.

[0048] Add 200 μL of bacterial solution to 10 mL of LB liquid medium containing 12.5 mg / L tetracycline resistance and culture at 42°C on a shaking platform.

[0049] 2. Prepare 2,4-DNT solution Prepare 2 mg / mL stock solution of 2,4-DNT (10 mg 2,4-DNT dissolved in 5 mL anhydrous ethanol); Prepare the diluted 2,4-DNT solution in the following proportions: 10 mg / L: 5 μL stock solution + 980 μL M9 medium + 15 μL anhydrous ethanol; 5 mg / L: 2.5 μL mother solution + 980 μL M9 medium + 17.5 μL anhydrous ethanol; 1 mg / L: 0.5 μL mother solution + 980 μL M9 medium + 19.5 μL anhydrous ethanol; 0 mg / L: 980 μL ASYE medium + 20 μL anhydrous ethanol.

[0050] The concentration of ethanol in each DNT solution was set to 2%.

[0051] 3. Add sample Take 90 μL of bacterial solution + 10 μL of 0 mg / L, 1 mg / L, 5 mg / L, 10 mg / L 2,4-DNT stock solution and add it to 1.5 ml EP tube, so that the final concentration of 2,4-DNT is 0 mg / L, 0.1 mg / L, 0.5 mg / L, 1 mg / L, respectively; take 99 μL of bacterial solution + 1 μL of 1 mg / L, 5 mg / L 2,4-DNT stock solution and add it to 1.5 ml EP tube, so that the final concentration of 2,4-DNT is 0.01 mg / L, 0.05 mg / L, respectively. Make 4 parallels for each concentration. Cultivate at 42°C.

[0052] 4. Luminescence detection Each sample was sampled once an hour for a total of 10 hours. The fluorescence intensity of the G1 strain was detected using an ELISA reader (Biotek).

[0053] 5. The mutated promoter PyqjfC55 in the G1 strain was sequenced by a biological company (Shanghai Bioengineering) and it was found that there were three base mutations compared to yqjfC55. Figure 4The nucleotide sequence of the mutated promoter PyqjfC55 is shown in SEQ ID NO.3.

[0054] The results are as follows Figure 3 , indicating that the G1 recombinant strain constructed by the present invention, as a biosensor for detecting explosive molecules under high temperature conditions, has different fluorescence intensities under the action of different concentrations of DNT. The higher the DNT concentration, the more obvious the detection effect. The fluorescence effect induced by the lowest 0.1 mg / L 2,4-DNT explosive molecule can form a gap with that without induction ( Figure 3 ). Moreover, at 42°C, it can stably and effectively detect explosive molecules.

[0055] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. An optimized promoter PyqjfC55, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

3.

2. Use of the optimized promoter PyqjfC55 described in claim 1 in the preparation of a high temperature resistant microbial sensor.

3. The use according to claim 2, characterized in that: The application comprises the following steps: (1) The yellow fluorescent protein EYFP and the promoter PyqjfC55 were amplified by PCR, and the two fragments EYFP and PyqjfC55 obtained by purification were connected with the plasmid pACYC. The ligation products were transformed into Escherichia coli competent cells, and positive clones were screened on LB solid plates containing antibiotics to obtain the recombinant plasmid pACYC-PyqjfC55-EYFP; (2) The recombinant plasmid pACYC-PyqjfC55-EYFP was transformed into competent Escherichia coli cells and screened on LB solid plates containing 12.5 μg / mL tetracycline resistance at 37°C. The positive clone obtained was the pACYC-C55-EYFP plasmid, which was then introduced into Escherichia coli cells to obtain an engineered strain of a high-temperature resistant microbial sensor containing the optimized promoter PyqjfC55.

4. The use according to claim 3, characterized in that: The mass ratio of the fragment EYFP and the fragment PyqjfC55 to the plasmid pACYC is 1:1:

5.

5. The use according to claim 3, characterized in that: The nucleotide sequence of the EYFP encoding gene is shown as SEQ ID NO.

2.

6. Use of the high temperature resistant microbial sensor prepared according to claim 3 to detect explosive molecules in a high temperature application environment.

7. The use according to claim 6, characterized in that: The method of using the microbial sensor is as follows: after mixing the engineered strain containing the high-temperature resistant biosensor with the sample to be tested, incubate at 40-42°C, take samples once every hour for a total of ten hours, use an enzyme marker to detect the fluorescence intensity value, and detect the concentration of the sample to be tested based on the fluorescence intensity.

8. The use according to claim 7, characterized in that: The sample to be tested is the explosive molecule 2,4-DNT, and the host of the engineered strain is Escherichia coli.

9. The use according to claim 8, characterized in that: The detection concentration range of the 2,4-DNT is 0.1 mg / L~10 mg / L.

10. The use according to claim 8, characterized in that: The high temperature application environment includes deserts, mountains and grasslands.

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